Method to stabilize cell suspensions using aged heavy metal solution and paraformaldehyde

ABSTRACT

A stabilized cell suspension is prepared by treating the cell suspension with a solution of a heavy metal compound, which has been aged at pH 6.5-7.5 prior to use to allow a precipitate to form, removing the precipitate from the heavy metal solution, mixing the aged heavy metal solution with the cell suspension to form a first mixture, and mixing the first mixture with a paraformaldehyde solution to form a stabilized cell suspension which is still capable of lysis.

BACKGROUND OF THE INVENTION

This invention relates to the preparation and stabilisation of cellsuspensions, and more particularly to a novel method for preparing andstabilising cell suspensions, and to the use of novel stabilised cellsuspension preparations in the quality control of analytical techniquessuch as UV microscopy and flow cytometric leucocyte immunophenotypingtechniques, immobilised antigen/antibody detection systems andhaematology analysers, and blood monitoring techniques such as zincprotoporphyrin (ZPP), red cell folate and blood glucose measurements.

UV microscopy and flow cytometry are techniques used in the diagnosis ofhaematological malignancies. They are also used to monitor the progressof patients infected with the Human Immunodeficiency Virus (HIV) whetherasymptomatic or suffering from ARC or full-blown AIDS. Quality control(QC) of these two techniques is extremely important to arrive at thecorrect diagnosis and to monitor effective therapeutic regimes. Thecurrent QC methods use freshly drawn blood or microspheres coated with afluorochrome.

The use of fresh blood on a daily basis fails to provide the informationon day to day variation of the technique or equipment. Furthermore,fluorochrome coated microspheres, though providing a day to day monitorof the flow cytometer's performance cannot be used for UV microscopywork. In addition, they cannot be used to provide quality control forthe labelling techniques of leucocytes.

Fixation of normal leucocytes, utilising compounds such asparaformaldehyde, although giving stability for 5-7 days increasescellular autofluorescence. This makes the preparation unsuitable for useas a long term quality control material. Furthermore, the lysis of redcalls by the whole blood lysing technique requires a preparation thatwill quality control this procedure. The current methods of fixing theleucocytes inhibit this lysing procedure resulting in a significantincrease in debris that interferes with the tests.

Other QC equipment requiring the use of whole blood samples or bloodproducts for calibration include haematology analysers where currentlyfixed blood from donkeys and turkeys is used because a suitable sourceof stabilised human blood is not available. Zinc protoporphyrin (ZPP)and red cell folate monitoring techniques also require a freshsuspension of red blood cells for calibration, again because a suitablestabilised source is not available. Finally the lack of a stabilisedsource of whole human blood for calibration purposes limits thepossibility for diabetics to carry out blood sugar monitoring at home.

In International Application No. WO 91/17436, the entire disclosure ofwhich is incorporated herein by reference, there is described a blooddiluent and lysing agent for differential determination of white bloodcells (leucocytes) in which the stabilising agent is diazolidinyl urea.Such leucocyte preparations have not been suggested as flow cytometricpreparations possibly because they have insufficient stability and lackcertain specific antigenic activity for those routine quality controlprocedures which need to assess-results from a large number oflaboratories.

International Application No. WO92/19951 discloses the use ofdiazolidinyl urea, imidazolidinyl urea,dimethylol-5,5-dimethylhydantoin, dimethylol urea,2-bromo-2-nitropropane-1,3-diol and quaternary adamantane as tissuefixatives which are free of aldehydes. The formulations may containinter alia mordants such as zinc, calcium, barium and chromium salts. Itis not suggested, however, that any of these salts have stabilisingproperties.

G81563839 provides a process for preparing a stabilised preparation oferythrocytes, wherein a suspension of erythrocytes in an aqueousisotonic solution is reacted with, simultaneously or successively in anysequence, an aliphatic aldehyde and a water-soluble salt of 2- or 3-valent chromium. In the examples, the erythrocytes are treated withformaldehyde and glutaraldehyde, followed by an aqueous solution ofchromium III chloride.

It has been found that cell suspensions containing erythrocytes treatedin this way are no longer suitable for use in a whole blood lysingtechnique, and thus the process of GB1563839 cannot be used to preparestabilised whole blood samples, for example, for flow cytometry qualitycontrol.

GB2001757 describes a method of preparing a stable suspension of bloodplatelets which comprises adding chromium ions to a platelet-rich plasmaand adding at least one organic aldehyde to the chromium-platelet-richplasma mixture. In the examples, the platelet-rich plasma is treatedwith solutions of potassium dichromate and glutaraldehyde. This process,if used on leucocyte cells, substantially increases autofluorescence andrenders the suspension unsuitable for use in flow cytometry.

The entire disclosures of the above-mentioned patent and applicationsare incorporated herein by reference.

It will be appreciated from the above, that there is a need for animproved method for stabilising cell suspensions, particularly of wholeblood and blood products, giving a cell preparation that is stillcapable of lysis, for a variety of quality control, monitoring andcalibration applications.

In UK Patent Application No. GB2279653 it is disclosed that certainheavy metal compounds can stabilise leucocyte preparations formed fromlysed whole blood, and that such stabilised preparations can be added toleucocyte-deplated blood to provide a stabilized whole blood preparationwith high antigenic activity. The entire disclosures of theseapplications are incorporated herein by reference.

OBJECTS OF THE INVENTION

It is an object of the invention to provide an improved stabilised cellsuspension preparation which is still capable of lysis and an improvedmethod for producing such stabilised cell suspensions.

It is also an object of the invention to provide a stabilised wholeblood preparation which is still capable of lysts and a method ofmanufacturing such a preparation.

It is a still further object of the invention to provide stable qualitycontrol materials which can be used in a wide spectrum of qualitycontrol, analysis and monitoring techniques.

SUMMARY OF THE INVENTION

It has now been discovered that a wide range of cell suspensionscomprising cells capable of lysis can be stabilised by the sequentialaddition of a heavy metal compound and paraformaldehyde to thesuspension in effective amounts, and that such stabilised cellsuspensions will remain active for much longer periods then those knownhitherto.

In one aspect the invention provides a method of treating a suspensionof Tells capable of lysis, to produced a stabilised cell suspensionstill capable of lysis, which comprises:

treating the cell suspension, in an aqueous medium maintained at a pH offrom 6.5 to 7.5, sequentially with,

1) an aged aqueous solution of a heavy metal compound, the pH of thesolution being from 6.5 to 7.5, and the solution having been maintainedat a pH of from 6.5 to 7.5 for a period of time sufficient to allow anyprecipitate to form, and

2) an aqueous solution of paraformaldehyde, the pH of the solution beingfrom 6.5 to 7.5.

The invention is particularly applicable to the stabilisation of wholeblood and of blood products and will be henceforth more particularlydescribed with reference thereto. It is to be understood, however thatthe invention is not limited to the stabilisation of such materials, andis broadly applicable to a wide range of cell suspension capable oflysis.

In a further aspect, therefore, the invention provides an unseparatedstabilised whole blood preparation capable of lysis in which thestabilizing agent comprises effective amounts of a heavy metal compound,particularly a heavy metal salt, and paraformaldehyde, and a method ofstabilising a whole blood preparation capable of lysis by addingeffective amounts of the compound and paraformaldehyde thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1b, 1c, and 1d show the flow cytometric characteristics of"fresh" blood after staining for the antigens CD3, CD4, CD8, and CD20;

FIGS. 2a, 2b, 2c, and 2d show the effect of ageing on the flowcytometric FSC, and SSC of "fresh" unstabilised blood, over an 8 dayperiod;

FIGS. 3a(i), 3b(i), 3a(ii), 3b(ii), 3a(iii), and 3b(iii) show the effectof ageing on the expression of antigens shown in FIGS. 1b, 1c, and 1d,over a 10 day period;

FIGS. 4a and 4b show the stability of FSC, SSC and the negative controlcharacteristics, as determined by flow cytometry, upon the stabilisedwhole blood preparation, over a 60 day period;

FIGS. 5a and 5b show the stability of various antigens as measured byflow cytometry;

FIG. 6 shows the effect of storage on the flow cytometric leucocytedifferential on "fresh" blood over an 8 day period;

FIG. 7 shows the stability of the flow cytometric differential of thestabilised blood preparation over a 25 day period;

FIG. 8 shows the stability in zinc protoporphyrin (ZPP) level in asample of the stabilised whole blood preparation over a 36 day period;

FIG. 9 shows the stability of the total white cell count and totallymphocyte count over a 117 day period for a sample of the stabilisedwhole blood preparations;

FIG. 10 shows the measurement of red cell folate for samples of thestabilised whole blood preparation and fresh stored whole blood over a50 day period;

FIGS. 11a and 11b show the stability of the FSC, SSC and negativecontrol characteristics, as determined by flow cytometry upon thestabilised whole blood preparation at 11 days post stabilisation;

FIGS. 12a, 12b, and 12c show the stability of the antigens CD3, CD4,CD8, and CD19, as measured by flow cytometry at 11 days poststabilisation;

FIGS. 13a and 13b show the stability of the FSC, SSC and negativecontrol characteristics as determined by flow cytometry, upon thestabilised whole blood preparation, at 29 days post stabilisation;

FIGS. 14a, 14b, and 14c show the stability of the antigens CD3, CD4,CD8, and CD19, as measured by flow cytometry at 29 days poststabilisation;

FIGS. 15a and 15b show the stability of FSC, SSC and negative controlcharacteristics, as determined by flow cytometry upon the stabilisedwhole blood preparation at 11 days post stabilisation;

FIGS. 16a, 16b, and 16c show the stability of the antigens CD3, CD4,CD8, and CD19, as measured by flow cytometry at 11 days poststabilisation;

FIGS. 17a and 17b show the stability of FSC, SSC and negative controlcharacteristics, as determined by flow cytometry upon the stabilisedwhole blood preparation at 29 days post stabilisation; and,

FIGS. 18a, 18b, and 18c show the stability of the antigens CD3, CD4,CD8, and CD19, as determined by flow cytometry at 29 days poststabilisation.

DETAILED DESCRIPTION OF THE INVENTION

In UK Patent Application No. GB2279653, leucocyte preparations separatedfrom lysed whole blood are stabilised by the addition of a stabilisingagent, and then added back to leucocyte depleted whole blood (red bloodcells) to from a stabilised whole blood preparation. The presentinvention provides an alternative method for the preparation ofstabilised whole blood in which the lysing and separating-steps can beomitted.

The invention will now be particularly described with reference to themanufacture of stabilised whole blood preparations for use in laboratoryquality control procedures, although it will be appreciated that theinvention is not limited thereto and that, for example, the stabilisedwhole blood preparations may find other uses, and that the stabilisationprocedures described may find application in the manufacture ofstabilised cell suspensions from sources other than blood, for examplemicrobial cells, plant cells and similar materials derived from livingtissue.

In this specification, a suspension of cells capable of lysis is definedas: a suspension of cells, such as, for example, whole human blood, inwhich specific cell populations can be selectively lysed usingerythrocyte lysing reagents. Lysis comprises rupture of the cellmembrane and leakage of the cell contents. Lysing reagents andconditions suitable for the selective lysis of the erythrocyte componentof whole human blood include, for example, treatment in aqueous solutionwith an ammonium salt, for example, ammonium chloride, or a quaternaryammonium salt and urea, or a substituted urea, or urea derivative, asdescribed in UK Patent Application No. GB92279653.

Whole blood preparations in this specification include preparationscontaining substantially all the components of fresh blood, andpreparations containing substantially all the components of fresh bloodother than plasma.

The process by which the stabilisation of the whole blood is achieveduses a series of stages incorporating the addition of both organic andinorganic compounds The initial process of drawing a unit of blood iswell documented and can be any of those used by those employed in theart of venesection. Anticoagulated blood is preferably used, and severalsuitable anticoagulants are commercially available. However, the use ofa potassium EDTA salt is preferred.

The process of stabilising the whole blood sample is usually performedwithin 24 hours of venesection but is preferably performed within 2hours.

The fresh whole blood, containing an anticoagulant, is firstcentrifuged, and the plasma removed and retained.

The remaining cells are washed and then treated with an aged aqueoussolution of a heavy metal compound, and particularly a heavy metal salt.Suitable heavy metals are those with complexing properties and having anatomic weight greater than 20, for example, aluminium, and transitionmetals such as chromium and manganese. Suitable metal compounds includeinorganic acid salts, and particularly chlorides.

Particularly good results have been obtained using chromium compounds,such as, for example, chromic salts,for example, chromic chloride CrCl₃,and aluminium compounds, for example aluminium chloride AlCl₃.

The heavy metal compound can be dissolved in the appropriateconcentration in an aqueous solution which is buffered to a pH of 6.5 to7.5, preferably 6.7 to 7.4, more preferably around 7.2, and aged for aperiod of time sufficient to allow any precipitate to form. Suitablebuffered aqueous solutions include, for example, phosphate bufferedsaline solutions.

Preferably, however, the heavy metal compound is stored as a relativelyconcentrated solution, for example, as a 1% w/v solution and is dilutedto the desired concentration level with an appropriate aqueous buffersolution before use. With the preferred heavy metal compounds, it isfound that the concentrated solutions are stable, but that on dilutionprecipitate may form as the pH rises. Sufficient time should be allowedfor the solution to age and precipitation to occur before the solutionis used.

The diluted aqueous solution of the heavy metal compound is thereforeallowed to stand, preferably for at least one week, more preferably fromone week to one month, before use. The reason why the performance of thesolution improves with standing is at understood, but may be due to theformation of hydrated metal hydroxy ionic species in the solution. Ithas been observed with some buffered solutions of chromium compounds,for example, that the freshly made solutions change from green to purpleover a 24 hour period, indicating the presence of charged complex ions,together with the formation of a precipitate which may be a chromiumhydroxy polymeric species. This is preferably filtered off from thesolution before use. The formation of a precipitate will, of course,lower the concentration of heavy metal ions in the solution, and if thisshould occur, an analysis of the solution should be carried out todetermine whether the concentration of the heavy metal compound is stillwithin the preferred range.

The aged solution of heavy metal compound retains its effectiveness overconsiderable periods of time, but is preferably discarded after 12months and more preferably after 6 months.

The aqueous solution of the heavy metal compound is preferably added tothe whole blood preparation as an isotonic solution in which the optimumfinal concentration of the heavy metal compound is preferably less than1% w/v. more preferably from 0.005% to 0.75% w/v, still more preferably0.01% to 0.5% w/v and most preferably from 0.05% to 0.25% w/v,. forexample, 0.1% w/v. The pH of the aqueous solution is preferably from 6.5to 7.5, preferably 6.7 to 7.4, before addition. The whole bloodpreparation is preferably exposed to the heavy metal compound for anincubation period of from 5 minutes to 18 hours, for example, about 60minutes.

The incubation temperature is preferably from 0° C. to 8° C., morepreferably from 2° C. to 60° C., for example about 4° C.

After the first incubation period, the whole blood is treated with anaqueous solution of paraformaldehyde, which can, for example, be at apreferred final concentration of up to 1% w/v, preferably from 0.1% to0.5% w/v, for example 0.35% w/v. When making up the paraformaldehydesolution it is preferable to keep the temperature below 60° C., morepreferably below 50° C., in order to avoid the reversion ofparaformaldehyde to formaldehyde and evolution of formic acid. The ageof the paraformaldehyde solution has also ben found to be important, andit is preferably greater than one week old, and no older than one month,preferably no older than two weeks.

In a preferred aspect of the invention, it has been found that very goodresults are obtained by mixing the paraformaldehyde, preferably inaqueous solution, with an aged aqueous solution of a heavy metalcompound.

The aged aqueous solution of a heavy metal compound may be any of thosepreviously described, and the metal compound may be present in the mixedsolution in an amount of from 0.01% to 0.5% w/v. Preferably the ratio ofheavy metal compound to paraformaldehyde in the solution is in the rangeof from 5:1 to 1:50, for example, around 1:3.5.

Preferably the aqueous paraformaldehyde solution has a pH of from 6.5 to7.5, more preferably 6.7 to 7.4, and may comprise a suitable isotonicbuffer. A preferred solution can be, for example, a 0.85% phosphatebuffered saline solution.

A preferred mixed paraformaldehyde/heavy metal compound solution can bemade, for example, by mixing a 0.7% w/v solution of paraformaldehydewith an equal volume of a 0.2% w/v solution of the heavy metal compound.

Exposure to the paraformaldehyde or mixed solution is preferably from 6hours to 24 hours at a temperature range of from 0° to 8° C., preferablyfrom 2° to 6° C., for example, about 4° C.

The interval between the sequential treatment stages 1) and 2) ispreferably at least 30 minutes, more preferably at least one hour, stillmore preferably at least 12 hours, for example 24 hours.

After washing in isotonic phosphate buffered saline solution the plasma,if previously removed, can be added back to the stabilised whole bloodpreparation. This plasma can, but need not necessarily be, that obtainedfrom the original donation. Bacterial growth inhibitors and antibioticsfor example gentamycin are preferably also added to the finalpreparation. The preparation is then retained at between 0° C. and 8° C.for from 1 to 5 days before use.

All the above procedures are preferably carried out under sterileconditions and preferably the entire procedure is carried out in thevenesection pack in which the donated blood is collected.

In the commercial manufacture of a stabilised whole blood preparationaccording to the invention, it is possible and may be preferable to poola number of units of donated blood from different sources and tostabilise the resultant pool.

In another aspect of the invention, it is possible to add further celllines to the whole blood preparation, either before or after thestabilisation of the whole blood preparation. For example, it ispossible to add additional CD34+ cells in the form of cell line KG1 oras human umbilical cord blood (which is rich in CD34+ cells) to normalwhole blood to provide a quality control for CD34+ cell measurements.

The stabilisation process can be applied to both normal and leukaemiccells providing a known normal control and an abnormal (leukaemic)control. It has been found that with normal cells, using chromiumchloride as the metal compound usually provides the best results.However with leukaemic cells, aluminium chloride often gives the bestresults.

The invention provides a method of stabilising whole human bloodpreparations without the necessity to lyse the red blood cells(erythrocytes) and stabilise the leucocytes separately.

The stabilised whole blood preparations of the invention, can provide anexcellent stable quality control and reference material, which can beused in leucocyte immunophenotyping by both UV microscopy and flowcytometry. The preparation is of value in the quality control of thewhole blood lysing procedure without any excess contamination fromdebris. The stabilised sample can comprise all of the normal peripheralblood leucocytes (granulocytes, monocytes and lymphocytes) or subsetsthereof. The procedure can, under optimum conditions, retain theleucocyte antigenic profiles ensuring phenotyping and quality control ofthe procedure. The values for the common antigenic determinants can bedetermined and can be stable for more than 300 days. The investigatorcan also successfully derive values for antigens that may be of specificinterest. Furthermore, the preparation can be used to quality controlthe differential obtained from the flow cytometer. This is a parameterthat is used for the monitoring of anti-viral therapy in HIV infectedindividuals.

New methods for phenotyping blood specimens are being developed which donot require the sample to be treated with a lysing agent after staining.These techniques are termed no-wash, no-lyse. The whole bloodpreparations of this invention can be used to provide quality controlfor these techniques and can also be used on flow cytometer that analyseno-wash, no-lyse procedures.

In general the stabilised whole blood preparations of the invention canbe used in quality control of UV microscopy and flow cytometricimmunophenotyping techniques, both of the whole blood lysis and wholeblood non-lysis techniques,

Investigations with preferred embodiments of the stabilised whole bloodpreparations of the invention show that they are also suitable for usein immunocytochemical analysis using techniques such as the alkalinephosphatase anti-alkaline phosphatase immunocytochemical technique(APAAP) and can for example be used to determine the antigenic profileof leucocytes on peripheral blood smears. They can be used as a day today reference materials or in eternal (inter-laboratory) qualitycontrol. Multiple smears can be made in advance and then stored at -20°C. until use. The smears can be stained on a daily basis, or used ascontrols when staining other smears.

The stabilised whole blood preparations of the invention may also findapplication as standard reference materials for use in enzyme linkedimmunosorbent assay techniques (ELISA) and in immunoradiometric assaytechniques.

Further uses of the stabilised whole blood preparations of the inventionin the laboratory may include as a quality control and calibrant forhaematology analyzers, as a quality control material for monitoring irondeficiency by the zinc protoporphyrin technique (ZPP), and as a qualitycontrol material in the red cell folate technique. Finally, on a broaderbasis, the stabilised whole blood preparations of the invention may findapplication in the quality control of blood glucose level tests, therebyenabling diabetic patients to carry out this technique in their ownhomes.

The invention is illustrated by the following Examples:

EXAMPLE 1

A stabilised whole blood preparation is made up as described below andits ageing characteristics compared with a similar untreated whole bloodsample.

Preparation

1. Venesect a unit of 500 ml of blood into a sterile venesection packcontaining 600 mg of ethylenediaminetetraacatic acid (disodium or tripotassium salt) dissolved in 50 ml of phosphate buffered saline solution(PBS).

2. Centrifuge the unit at 800 g for 1 hour. Remove and retain theplasma.

3. Wash the remaining cells twice with sterile phosphate buffered salinesolution (PBS)

4. Remove the supernatant PBS.

5. Add 300 ml of filtered 0.1% aged (>1 month) Chromium Chloridehexahydrate (pH 6.7) in PBS and incubate for 1 hour in the dark at 4° C.mixing occasionally.

6. Centrifuge for 45 minutes at 800 g and decant supernatant.

7. Resuspend in 300 ml aged (>1 month) and filtered 0.1% ChromiumChloride hexahydrate pH 6.7 in 0.35% paraformaldehyde in PBS. Incubateat 4° C. in the dark for 16-22 hours.

8. Centrifuge at 800 g for 45 minutes and decant supernatant.

9. Wash twice by centrifuging at 800 g with PBS. Decant finalsupernatant.

10. Resuspend the stabilised whole blood in the plasma detailed in step2. Add broad range antibiotics such as gentamycin, ciprofloxacin.

12. Place at 4° C. for 2-3 days until use.

Phosphate buffered Saline pH 7.2

7.83 g/l Sodium Chloride

0.36 g/l disodium EDTA

0.28 g/l Potassium Chloride

0.26 g/l Potassium dihydrogen phosphate (monobasic)

2.35 g/l disodium hydrogen phosphate (dibasic)

The comparative results are illustrated in the accompanying Drawings inwhich:

FIG. 1 shows the flow cytometric characteristics of "fresh" blood afterstaining for the antigens CD3, CD4, CD8, and CD20. The staining wascarried out employing the whole blood lysis technique and the positivelevels related to the negative controls (a) Forward & side scatter (FSC& SSC) characteristics, (b) CD3 PE & CD20 FITC, (c) CD3 FITC & CD4 PE,(d) CD3 FITC & CD8 PE.

FIG. 2 shows the effect of ageing on the flow cytometric FSC, and SSC of"fresh" unstabilized blood over a period of 8 days (a) Day 1, (b) Day 2(c) Day 3, (d) Day 8.

FIGS. 3a and 3b show the effect of ageing on the expression of theantigens described in FIG. 1b, c & d over a 10 day period 3a (i) (ii)(iii) Day 1, and 3b (i) (ii) (iii) Day 10.

FIGS. 4a and 4b show the stability of FSC, SSC and the negative controlcharacteristics, as determined by flow cytometry, upon the stabilisedwhole blood preparation over a period of 60 days, 4a day 3, 4b day 60.

FIGS. 5a and 5b show the stability of the antigens CD3, CD4, CD8, CD19,and CD16+CD56, as measured by flow cytometry, over a 57 day period 5aDay 3, 5b Day 60.

FIG. 6 shows the effect of storage on the flow cytometric leucocytedifferential on "fresh" blood over a period of 8 days. Further analysisafter day 8 was not possible due to marked deterioration of the sample.Analysis used a FACScan flow cytometer.

FIG. 7 shows the stability of the flow cytometric differential of thestabilised blood preparation over a 25 day period. Analysis used aFACScan (trademark) flow cytometer.

FIG. 8 shows the stability in zinc protoporphyrin (ZPP) level in asample of the stabilised whole blood preparation over a period of 36days measured in μmoles ZPP/mole of haemoglobin.

FIG. 9 shows the stability of the total white cell count and totallymphocyte count over a 117 day period for a sample of the stabilisedwhole blood preparations measured using a Toa (Sysmex) NE8000(trademark) haematology analyser.

Finally, FIG. 10 shows the measurement of red cell folate for samples ofthe stabilised whole blood preparation and fresh stored whole blood overa 50-day period.

The flow Metric profile in FIG. 1 shows the normal position of thelymphocytes, monocytes and granulocytes after lysis of the red cells.The antigen staining characteristics are also shown in FIG. 1b, c, d, &e. As the sample increases with age (FIG. 2) the flow cytometric andantigen expression characteristics alter. At day 8 the amount of debrismakes the analysis unsatisfactory. The fluorescence labellingcharacteristics have also deteriorated as shown by FIG. 3.

FIG. 4 displays the forward and side scatter characteristics togetherwith the negative control of the stabilised sample at day 3 and day 60.The individual populations are retained in their respective positionsimmediately post stabilisation. After 60 days preservation the forwardand side scatter flow cytometric characteristics remain intact (FIG.4b). In addition the antigen expression characteristics remain unalteredover this period (FIG. 5). The same machine settings were retainedthroughout.

The stabilised blood preparation can be used to monitor the leucocytedifferential. Using a specific combination of antibodies directedagainst the CD14 and CD45 antigens a three-population differential canbe generated. FIG. 7 shows the stability of this parameter over 25 days,whereas FIG. 6 shows the instability of the "fresh" whole blood over an8 day period. Analysis of the latter after this time was aborted due tocontamination with excessive debris.

As shown in FIG. 8, ZPP levels in the stabilised whole blood preparationremain substantially constant, with only a very slight increase after 36days. Similarly, as shown in FIG. 9, the total white cell count andtotal lymphocyte count are also scarcely affected by time over a 117 dayperiod.

FIG. 10 shows the very substantial improvement obtained in the stabilityof red cell folate using a stabilised whole blood preparation accordingto the invention.

EXAMPLE 2

A stabilised whole blood preparation is made up as described in Example1, except that the reactions with chromium chloride hexahydrate and withthe mixture of chromium chloride hexahydrate and paraformaldehyde arecarried out in phosphate buffered saline solution at a pH of 7.2.

FIG. 11 shows the stability of the FSC, SSC (FIG. 11a) and negativecontrol (FIG. 11b) characteristics, as determined by flow cytometry uponthe stabilised whole blood preparation, at 11 days post stabilisation.

FIG. 12 (a), (b) and (c) shows the stability of the antigens CD3, CD4,CD8, and CD19 as measured by flow cytometry at 11 days poststabilisation.

FIG. 13 (a) and (b) shows the stability of the FSC, SSC and negativecontrol characteristics as determined by flow cytometry, upon thestabilised whole blood preparation, at 29 days post stabilisation.

FIG. 14 (a), (b) and (c) shows the stability of the antigens CD3, CD4,CD8 and CD19 as measured by flow cytometry at 29 days poststabilisation.

EXAMPLE 3

A stabilised whole blood preparation is made up as described in Example1, except that aluminium chloride hexahydrate is substituted for thechromium chloride hexahydrate, and the reactions with the aluminiumchloride and the mixture of aluminium chloride and paraformaldehyde arecarried out at pH 7.2.

FIG. 15 (a) and (b) shows the stability of FSC, SSC and negative controlcharacteristics, as determined by flow cytometry upon the stabilisedwhole blood preparation at 11 days post stabilisation.

FIG. 16 (a), (b) and (c) show the stability of the antigens CD3, CD4,CD8 and CD19 as measured by flow cytometry at 11 days poststabilisation.

FIG. 17 (a) and (b) shows the stability of FSC, SSC and negative controlcharacteristics as determined by flow cytometry upon the stabilisedwhole blood preparation at 29 days post stabilisation.

FIG. 18 (a), (b) and (c) shows the stability of the antigens CD3, CD4,CD8 and CD19 as determined by flow cytometry at 29 days poststabilisation.

The results show very good stabilisation of the whole blood preparation,with very little debris even after 29 days and good retention ofantigenic characteristics.

The reader's attention is directed to all papers and documents which arefiled concurrently with this specification and which are open to publicinspection with this specification, and the contents of all such papersand documents are incorporated herein by reference.

All the features disclosed in this specification (including anyaccompanying claims, abstract and drawings), and/or all of the steps orany method or process so disclosed, may be combined in any combination,except combinations where at least acme of such features and/or stepsare mutually exclusive.

Each feature disclosed in this specification (including any accompanyingclaims, abstract and drawings), may be replaced by alternative featuresserving the same, equivalent or similar purpose, unless expressly statedotherwise. Thus, unless expressly stated otherwise, each featuredisclosed is one example only of a generic series of equivalent orsimilar features.

The invention is not restricted to the details of the foregoingembodiment(s). This invention extends to any novel one, or any novelcombination, of the features disclosed in this specification (includingany accompanying claims, abstract and drawings), or to any novel one, orany novel combination, of the steps of any method or process sodisclosed.

We claim:
 1. A method of treating a suspension of cells capable of lysiscomprising the steps of:preparing an aqueous solution of a heavy metalcompound which has been aged at a pH of from 6.5 to 7.5 for at least 24hours to allow precipitate to form; removing the precipitate from thesolution to form an aged aqueous solution of the heavy metal compound,the aged aqueous solution comprising less than 1% weight per volume(w/v) of the heavy metal compound; mixing the cell suspension and theaged solution of the heavy metal compound at a pH of from 6.5 to 7.5 toform a first mixture; and, mixing the first mixture with aparaformaldehyde solution comprising up to 1% weight per volume (w/v)paraformaldehyde at a pH of from 6.5 to 7.5 to form a stabilised cellsuspension which is still capable of lysis.
 2. The method according toclaim 1, in which the cell suspension is a whole blood preparation. 3.The method according to claim 1 or 2, in which the heavy metal isaluminium or a transition metal.
 4. The method according to claim 1, inwhich the heavy metal compound is a metal salt.
 5. The method accordingto claim 4, in which the metal salt is chromic chloride.
 6. The methodaccording to claim 4, in which the metal salt is aluminum chloride. 7.The method according to claim 1, in which the aqueous solution of aheavy metal compound has a pH of from 6.7 to 7.4.
 8. The methodaccording to claim 1, wherein the step of preparing the aqueous solutionof the heavy metal compound further comprises the step of forming theaqueous solution of the heavy metal compound by diluting a relativelyconcentrated solution of the heavy metal compound and raising its pHfrom 6.5 to 7.5 by the addition of an aqueous buffer solution.
 9. Themethod according to claim 1, in which the aqueous solution of a heavymetal compound is aged for at least one week before use.
 10. The methodaccording to claim 1, in which the aqueous solution of the heavy metalcompound is added to the cell suspension as a 0.01% to 0.5% w/vsolution.
 11. The method according to claim 1, in which the cells areexposed to the aqueous solution of the heavy metal compound for a periodof from 5 minutes to 18 hours at a temperature of from 0° C. to 8° C.12. The method according to claim 1, in which the paraformaldehyde isadded as a 0.1% to 0.5% w/v aqueous solution.
 13. The method accordingclaim 1, in which the paraformaldehyde solution is aqueous and has a pHof from 6.7 to 7.4.
 14. The method according to claim 1, wherein in thestep of mixing the first mixture with the paraformaldehyde solution, theparaformaldehyde solution further comprises an additional amount of anaged solution of a heavy metal compound having a pH in the range of from6.5 to 7.5.
 15. The method according to claim 14, in which the ratio ofheavy metal compound in the additional amount of the aged solution ofthe heavy metal compound to paraformaldehyde in the solution is in therange of from 5:1 to 1:50.
 16. The method according to claim 1, whereinin the step of mixing the first mixture with the paraformaldehydesolution, the exposure of the cell suspension of the first mixture tothe paraformaldehyde solution is for a time period from 6 to 24 hours ata temperature of from 0° C. to 8° C.
 17. The method according to claim1, in which the steps are carried out sequentially at a time interval offrom 1 hour to 24 hours.
 18. The method according to claim 1, in which afurther cell line is added to a whole blood preparation, either beforeor after the stabilisation of the whole blood preparation.
 19. Themethod according to claim 18, in which the cell line comprises CD34+cells.
 20. A stabilised cell suspension capable of lysis prepared by amethod according to claim
 1. 21. The method of claim 1 wherein theparaformaldehyde solution further comprises an additional amount of anaged solution of a heavy metal compound.
 22. A method of treating asuspension of cells capable of lysis comprising the steps of:preparing a0.1%-0.5% aqueous solution of a heavy metal compound which has been agedat pH 6.5 to 7.5 for at least one week, which is an amount of timesufficient to allow any precipitate to form; removing the precipitatefrom the solution to form an aged aqueous solution of the heavy metalcompound; mixing the cell suspension and the aged solution of the heavymetal compound at pH 6.5 to 7.5 and at a temperature of 0° C. to 8° C.to form a first mixture; and, mixing the first mixture with a solutioncomprising 0.1%-0.5% paraformaldehyde at a pH of 6.5 to 7.5 and at atemperature of 0° C. to 8° C. to form a stabilized cell suspension whichis still capable of lysis.
 23. The method of claim 22 wherein theparaformaldehyde solution further comprises the aged solution of a heavymetal compound.